The invention relates to immunological reagents for diagnostic and therapeutic use in colon and other cancers. In particular, the invention relates to humanized anti-carcinoembryonic antigen (xe2x80x9cCEAxe2x80x9d) monoclonal antibodies (xe2x80x9cmAbsxe2x80x9d) that have the binding affinity characteristics of corresponding mouse anti-CEA mAb (MN14) and the antigenic and effector properties of a human antibody. Further, the invention relates to humanized mAbs in which the complementarity determining regions (xe2x80x9cCDRsxe2x80x9d) of an anti-CEA murine mAb is grafted into the framework regions of a human antibody, to DNAs that encode such CDR-grafted antibodies, to vectors and transformed hosts for propagating and expressing the DNAs, and to conjugates of the antibodies useful in diagnostic and therapeutic applications.
A promising approach to cancer diagnosis and therapy involves the use of targeting antibodies to deliver diagnostic and therapeutic agents directly to the site of a malignancy. Over the past decade, a wide variety of tumor-specific antibodies and antibody fragments have been developed, as have methods to conjugate the antibodies to drugs, toxins, radionuclides or other agents, and to administer the conjugates to patients. These efforts have produced great progress, but a variety of largely unanticipated problems have limited the diagnostic and therapeutic utility of some of the reagents thus far developed.
Among the most intractable problems is that which is caused by the human immune system itself, which may respond to the targeting conjugate as a foreign antigen. For instance, patients treated with drugs or radionuclides complexed with murine monoclonal antibodies (which have been the most commonly used targeting antibodies for human) develop circulating human anti-mouse antibodies (HAMAs) and a generalized immediate type-III hypersensitivity reaction to the antibody moiety of the conjugate. Furthermore, even when adverse side effects are minimal (for example, as in a single administration), circulating HAMAs decrease the effective concentration of the targeting agent in the patient and therefore limiting the diagnostic or therapeutic agent from reaching the target site.
Several approaches have been developed to overcome or avoid this problem, with only limited success. One strategy has been to chemically modify the targeting antibody to suppress its antigenicity. For example, conjugation of polyethylene glycol to the targeting antibody (PEGylation) is reported to reduce antigenicity of antibodies. Another approach has been to characterize the situs of antigenicity in an antibody and then remove it. In this vein, Fabxe2x80x2, F(ab)2 and other antibody fragments have been used in place of whole IgG. In addition, attempts have been made to reduce the adverse effects of HAMA by plasmaphoretically removing HAMA from blood. Immunosuppressive techniques also have been used to ameliorate the adverse effect of the foreign antibody sufficiently to permit multiple treatments with the targeting agent.
None of these approaches has proven altogether satisfactory. An important need persists for a means to reduce or eliminate the adverse immune response to targeting antibody and antibody conjugates in order to gain the full benefit of these diagnostic and therapeutic agents.
This goal has been achieved with the CDR-grafted humanized murine anti-human CEA mAbs that are described below.
It is an object of the present invention to provide a humanized Class III anti-CEA mAb in which the CDRs of a murine Class III anti-CEA mAb (MN14) are functionally engrafted to the amino acid sequence of a human antibody or antibody fragment to provide an immunological reagent with the anti-CEA binding properties of the murine Class III, anti-CEA mAb and the immunogenic properties of a human mAb in a human patient.
It is another object of the present invention to provide DNA constructs encoding such antibodies. Particular objects in this regard are substrate DNAs that facilitate genetic manipulation to produce improved antibodies and DNAs encoding the antibodies with advantageous properties in cell culture and antibody production.
Yet another object of the invention is to provide vectors for propagating the DNA and for expressing the antibody. A related object of the invention is to provide cells containing a vector for the purposes of storage, propagation, antibody production and therapeutic applications.
Still another object of the invention is to provide compositions comprising the antibodies for use in diagnosis and therapy. In this regard it is an object of the invention to provide conjugates comprising the antibodies complexed with imaging agents and therapeutic agents for ex vivo and in vivo imaging, diagnosis, prognosis and therapy, among others.
In accomplishing the foregoing objects, there has been provided, in accordance with one aspect of the present invention, a humanized mouse mAb, comprising the CDRs of a murine Class III, anti-CEA mAb (MN-14) engrafted to the framework regions of a heterologous (human) antibody, wherein the thus humanized mAb antibody retains the Class III, anti-CEA binding specificity of the murine mAb but in the patient is less immunogenic than is the parent MN-14 murine monoclonal antibody.
In a highly preferred embodiment, the light chain variable regions of the humanized antibody are characterized by the formula:
xe2x80x83FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4
wherein each FR is separately a framework region of a human antibody, and each CDR is separately in a complementarity-determining region of the light chains of MN-14, and the subscripts refer to light (xe2x80x9cLxe2x80x9d) chain regions. The heavy chain variable regions are characterized by the formula:
FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4
wherein FR and CDR have the same meanings as above, and wherein the subscripts xe2x80x9cHxe2x80x9d refer to heavy chain regions.
In one embodiment, CDRL1, has the amino acid sequence KASQD VGTSVA (SEQ. ID NO. 20); CDRL2 has the amino acid sequence WTSTR HT (SEQ. ID NO. 21); CDRL3 has the amino acid sequence QQYSL YRS (SEQ. ID NO. 22); CDRH1 has the amino acid sequence TYWMS (SEQ. ID. NO. 23); CDRH2 has the amino acid sequence EIHP DSSTI NYAPS LKD (SEQ. ID NO. 24); and, CDRH3 has the amino acid sequence LYFGF PWFAY (SEQ. ID NO. 25).
In another embodiment, FRL, has the amino acid sequence DIQLT QSPSS LSASV GDRVT ITC (SEQ. ID NO. 26); FRL2 has the amino acid sequence WYQQK PGKAP KLLIY (SEQ. ID NO. 27); FRL3 has the amino acid sequence GVP(S or D)RF SGS(G or V)S GTDFT FTISS LQPED IATYY C (SEQ. ID NO. 28); FRL4 has the amino acid sequence FGQGT KVEIK (SEQ. ID NO. 29); FRH1 has the amino acid sequence EVQLV ESGGG VVQPG RSLRL SCSSS GFDFT (SEQ. ID NO. 30), EVQLV ESGGG VVQPG RSLRL SCSAS GFDFT (SEQ. ID NO. 31), or QVQLQ ESGPG LVRPS QTLSL TCTSS GFDFT (SEQ. ID NO. 32); FRH2 has the amino acid sequence WVRQA PGKGL EWVA (SEQ. ID NO. 33), WVRQA PGKGL EWIA (SEQ. ID NO. 34), or WVRQP PGRGL EWIA (SEQ. ID NO. 35); FRH3 has the amino acid sequence RFTIS RDNSK NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 36), RFTIS RDNAK NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 37), or RVTML RDTSK NGSFL RLSSV TAADT AVYYC AS (SEQ. ID NO. 38); and FRH4 has the amino acid sequence WGQGT PVIVS S (SEQ. ID NO. 39), or WGQGT TVTVS S (SEQ. ID NO. 40); and wherein C may be in the sulfhydryl or disulfide form.
Another preferred embodiment comprises a diagnostic or therapeutic agent complexed to Class III, anti-CEA humanized mAb in which the CDRs of the antibody are derived from those of the MN-14 murine mAb and the FRs are derived from those of the heterologous (human) antibody, wherein the conjugate retains the Class III, anti-CEA binding specificity of MN-14, but is in humans less immunogenic than is murine MN-14. In one such embodiment the light chain and heavy chain variable regions are characterized as shown above and have amino acid sequences also as described above.
In yet another preferred embodiment, a method for diagnosing or treating a patient comprises the step of administering in an appropriate regimen the conjugate of the previous preferred embodiment.
Another preferred embodiment comprises an isolated, purified DNA that encodes the light chain, the heavy chain or both chains of the humanized antibody described above.
Another preferred embodiment comprises the DNA sequence of the CDRs and FRs described above.
Other objects, features and advantages of the present invention will become apparent from the following detailed description and appended claims.
The following terms or abbreviations are used in the present application. The meanings set out in this glossary are for illustrative purposes only. The full meaning of the terms will be apparent to those of skill in the art.
xe2x80x9cCDRxe2x80x9d is used as an abbreviation for Complementarity Determining Region. These are the regions within the variable regions of an antibody that are primarily, but not exclusively, responsible for antigen-antibody binding.
xe2x80x9cFRxe2x80x9d is an abbreviation for Framework Region. Broadly speaking, these are the portions of the variable regions of an antibody which lie adjacent to or flank the CDRs. In general, these regions have more of a structural function that affects the conformation of the variable region and are less directly responsible for the specific binding of antigen to antibody, although, nonetheless, the framework regions can affect the interaction.
xe2x80x9cChimericxe2x80x9d refers to an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from an antibody from a heterologous (other) species.
xe2x80x9cHumanizedxe2x80x9d refers to a chimeric antibody as defined above, but one in which the FR variable regions are derived from a human antibody.
xe2x80x9cHAMAxe2x80x9d refers to human antibodies directed to a mouse antibody, that are produced when a mouse antibody is administered to a human subject.
xe2x80x9cHAHAxe2x80x9d refers to human antibodies directed to a humanized mouse antibody.
xe2x80x9cCEAxe2x80x9d refers to carcinoembryonic antigen, a 180 kDa glycoprotein that is expressed in most adenocarcinomas of endodermally-derived digestive system epithelia and in some other cancers such as breast cancer and non-small cell lung cancer.
The letter xe2x80x9chxe2x80x9d as a prefix means xe2x80x9chumanizedxe2x80x9d.
Other abbreviations are used in accordance with Roitt et al., IMMUNOLOGY, 3rd ed. Mosby Year Book Europe Ltd. (1993), the entirety of which is herein incorporated by reference.
These and other terms used in the present disclosure are used in the same sense as ordinarily they are employed in the arts to which this invention pertains.